Master equation modeling of blackbody infrared radiative dissociation (BIRD) of hydrated peroxycarbonate radical anions.

Molecular cluster ions, which are stored in an electromagnetic trap under ultra-high vacuum conditions, undergo blackbody infrared radiative dissociation (BIRD). This process can be simulated with master equation modeling (MEM), predicting temperature-dependent dissociation rate constants, which are very sensitive to the dissociation energy. We have recently introduced a multiple-well approach for master equation modeling, where several low-lying isomers are taken into account. Here, we experimentally measure the BIRD of CO4●-(H2O)1,2 and model the results with a slightly modified multiple-well MEM. In the experiment, we exclusively observe loss of water from CO4●-(H2O), while the BIRD of CO4●-(H2O)2 leads predominantly to loss of carbon dioxide, with water loss occurring to a lesser extent. The MEM of two competing reactions requires empirical scaling factors for infrared intensities and the sum of states of the loose transition states employed in the calculation of unimolecular rate constants so that the simulated branching ratio matches the experiment. The experimentally derived binding energies are ΔH0(CO4●--H2O) = 45 ± 3 kJ/mol, ΔH0(CO4●-(H2O)-H2O) = 41 ± 3 kJ/mol, and ΔH0(CO2-O2●-(H2O)2) = 37 ± 3 kJ/mol. Quantum chemical calculations on the CCSD(T)/aug-cc-pVTZ//CCSD/aug-cc-pVDZ level, corrected for the basis set superposition error, yield binding energies that are 2-5 kJ/mol higher than experiment, within error limits of both experiment and theory. The relative activation energies for the two competing loss channels are as well fully consistent with theory.

Spotlight on CYP4B1.

The mammalian cytochrome P450 monooxygenase CYP4B1 can bioactivate a wide range of xenobiotics, such as its defining/hallmark substrate 4-ipomeanol leading to tissue-specific toxicities. Similar to other members of the CYP4 family, CYP4B1 has the ability to hydroxylate fatty acids and fatty alcohols. Structural insights into the enigmatic role of CYP4B1 with functions in both, xenobiotic and endobiotic metabolism, as well as its unusual heme-binding characteristics are now possible by the recently solved crystal structures of native rabbit CYP4B1 and the p.E310A variant. Importantly, CYP4B1 does not play a major role in hepatic P450-catalyzed phase I drug metabolism due to its predominant extra-hepatic expression, mainly in the lung. In addition, no catalytic activity of human CYP4B1 has been observed owing to a unique substitution of an evolutionary strongly conserved proline 427 to serine. Nevertheless, association of CYP4B1 expression patterns with various cancers and potential roles in cancer development have been reported for the human enzyme. This review will summarize the current status of CYP4B1 research with a spotlight on its roles in the metabolism of endogenous and exogenous compounds, structural properties, and cancer association, as well as its potential application in suicide gene approaches for targeted cancer therapy.

Differential Multimolecule Fingerprint for Similarity Search─Making Use of Active and Inactive Compound Sets in Virtual Screening.

In conventional fingerprint methods, the similarity between two molecules is calculated using the Tanimoto index as a numerical criterion. Thus, the query molecules in virtual screening should be most representative of the wanted compound class at hand. In the concept introduced here, all available active molecules form a multimolecule fingerprint in which the appearing features are weighted according to their respective frequency. The features of inactive molecules are treated likewise and the resulting values are subtracted from those of the active ones. The obtained differential multimolecule fingerprint (DMMFP) is thus specific for the respective class of compounds. To account for the noninteger representation within this fingerprint, a modified Sørensen-Dice coefficient is used to compute the similarity. Potentially active molecules yield positive scores, whereas presumably inactive ones are denoted by negative values. The concept was applied to Angiotensin-converting enzyme (ACE) inhibitors, ß2-adrenoceptor ligands, leukotriene A4 hydrolase inhibitors, dopamine D3 antagonists, and cytochrome CYP2C9 substrates, for which experimental binding affinities are known and was tested against decoys from DUD-E and a further background database consisting of molecules from the dark chemical matter, which comprises compounds that appear as frequent hitters across multiple assays. Using the 166 publicly available keys of the MACCS fingerprint and the larger PubChem fingerprint, actives were recovered with very high sensitivity. Furthermore, three marketed ACE inhibitors as well as the carbonic anhydrase II inhibitor dorzolamide were detected in the dark chemical matter data set. For comparison, the DMMFP was also used with a Bayesian classifier, for which the specificity (correctly classified inactives) and likewise the accuracy was superior. Conversely, the similarity score produced by the Sørensen-Dice coefficient showed its potential for the early recognition of (potentially) active molecules.

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering.

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L-1l-lysine from mannitol at a yield of 0.26 mol mol-1 and a maximum productivity of 2.1 g L-1 h-1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol-1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol-1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.

Highly regio- and stereoselective hydroxylation of vitamin D2 by CYP109E1.

Vitamin D2 is a form of vitamin D derived from mushrooms and plants which is structurally modified in the body due to the action of several enzymes. The resulting metabolites represent important compounds with potential bioactive properties. However, they are poorly studied and their availability is mostly limited. In order to identify new enzymes capable of producing vitamin D2 metabolites, we investigated a bacterial P450 monooxygenase, CYP109E1, which was previously shown to be a vitamin D3 hydroxylase. It was found that CYP109E1 catalyzes a vitamin D2 two-step hydroxylation at positions C24 and C25 resulting in the generation of 24(R),25-diOH VD2. Interestingly, the enzyme showed high selectivity towards vitamin D2, whereas it showed an unselective product pattern for the structurally similar vitamin D3. Our docking results for vitamin D2 and D3 revealed favorable hydroxylation positions for both substrates and suggested an explanation for the high selectivity of CYP109E1 towards vitamin D2. In addition, we established a whole-cell biocatalyst expressing CYP109E1 in Bacillus megaterium to produce 24(R),25-diOH VD2 and a production yield of 12.3 ± 1.2 mg/L was obtained after 48 h. To the best of our knowledge, this is the first report on the generation of 24(R),25-diOH VD2 by a microbial biocatalyst allowing a low-cost and eco-friendly production of this pharmaceutically interesting and expensive metabolite from the relatively cheap substrate, VD2.

Novel insights into oxidation of fatty acids and fatty alcohols by cytochrome P450 monooxygenase CYP4B1.

CYP4B1 is an enigmatic mammalian cytochrome P450 monooxygenase acting at the interface between xenobiotic and endobiotic metabolism. A prominent CYP4B1 substrate is the furan pro-toxin 4-ipomeanol (IPO). Our recent investigation on metabolism of IPO related compounds that maintain the furan functionality of IPO while replacing its alcohol group with alkyl chains of varying structure and length revealed that, in addition to cytotoxic reactive metabolite formation (resulting from furan activation) non-cytotoxic ω-hydroxylation at the alkyl chain can also occur. We hypothesized that substrate reorientations may happen in the active site of CYP4B1. These findings prompted us to re-investigate oxidation of unsaturated fatty acids and fatty alcohols with C9-C16 carbon chain length by CYP4B1. Strikingly, we found that besides the previously reported ω- and ω-1-hydroxylations, CYP4B1 is also capable of α-, ß-, γ-, and δ-fatty acid hydroxylation. In contrast, fatty alcohols of the same chain length are exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking results for the corresponding CYP4B1-substrate complexes revealed that fatty acids can adopt U-shaped bonding conformations, such that carbon atoms in both arms may approach the heme-iron. Quantum chemical estimates of activation energies of the hydrogen radical abstraction by the reactive compound 1 as well as electron densities of the substrate orbitals led to the conclusion that fatty acid and fatty alcohol oxidations by CYP4B1 are kinetically controlled reactions.

Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli.

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.

CYP109E1 from Bacillus megaterium Acts as a 24- and 25-Hydroxylase for Cholesterol.

In this study, the ability of CYP109E1 from Bacillus megaterium DSM319 to metabolize cholesterol was investigated. This steroid was identified as a new substrate to be converted by CYP109E1 with adrenodoxin and adrenodoxin reductase as redox partners in vitro. The biotransformation was successfully reproduced in vivo by using Bacillus megaterium cells that overexpressed CYP109E1. To enhance the production of cholesterol derivatives, an Escherichia coli based whole-cell system that harbored CYP109E1 was established. This novel system showed a 3.3-fold higher activity than that of the B. megaterium system, yielding about 45 mg L-1 of these products. Finally, the reaction products were isolated and identified to be the highly important cholesterol derivatives 24(S)- and 25-hydroxycholesterol.

Conservation and Relevance of Pharmacophore Point Types.

Pharmacophore models in general use a variety of features for distinct chemical characteristics, such as hydrogen-bond properties, lipohilicity, and ionizability. Usually, features have to match onto their identical type. To clarify if this stringent one-to-one assignment is justified, we investigated a set of 581 unique ligands from the BindingDB with known orientation inside the respective binding pockets and conducted a statistical analysis of the likelihood of observed exchanges in between the pharmacophore features, respectively their degree of conservation. To find out if certain features are obsolete, we derived a ranking to determine the most relevant ones. We found that the most conserved one-to-one feature is the negative ionizable (acids), followed by hydrogen-bond donor, positive ionizable (basic nitrogens), hydrogen-bond acceptor, aromatic, nonaromatic π-systems, and other lipophilic characteristics. The most likely exchanges were found between carboxylate groups and hydrogen-bond acceptors and likewise between basic nitrogens and hydrogen-bond donors, which reflects the characteristics of Lewis acids and bases. Exchanges between hydrogen-bond donors and hydrogen-bond acceptors are hardly more likely than by chance. The kind of target (e.g., kinase, phosphatase, protease, phosphodiesterase, nuclear receptor, metal-containing, or transmembrane protein) did not show substantial influence on the degree of conservation. The relevance of the actual pharmacophore features was found to be strongly dependent on the applied ranking scheme. Mutual information ranks all hydrophobic features as least important, whereas the aromatic feature is put into second place by using a geometric series. Both ranking schemes see the negative ionizable feature of higher significance than the positively ionizable feature.

Crystal Structure of CYP106A2 in Substrate-Free and Substrate-Bound Form.

CYP106A2 from Bacillus megaterium ATCC 13368 is known as a bacterial steroid hydroxylase that is also capable of hydroxylating a variety of terpenoids. To analyze the substrate specificity of this enzyme further, different resin acids of the abietane and pimarane types were tested with regard to binding and conversion. Product formation could be shown for all tested substrates. Spectroscopic studies revealed type I binding spectra for isopimaric acid, but dehydroabietic acid did not induce a high-spin shift of the enzyme. Interestingly, binding of abietic acid resulted in a type II difference spectrum typical for nitrogenous inhibitors. Co-crystallization of CYP106A2 with abietic acid and structure determination revealed bending of the heme cofactor when abietic acid was bound in the active site. Quantum chemical calculations strongly suggest that this heme distortion is the cause of the unusual spectroscopic characteristics.

Regioselective Acetylation of C21 Hydroxysteroids by the Bacterial Chloramphenicol Acetyltransferase I.

Chloramphenicol acetyltransferase I (CATI) detoxifies the antibiotic chloramphenicol and confers a corresponding resistance to bacteria. In this study we identified this enzyme as a steroid acetyltransferase and designed a new and efficient Escherichia-coli-based biocatalyst for the regioselective acetylation of C21 hydroxy groups in steroids of pharmaceutical interest. The cells carried a recombinant catI gene controlled by a constitutive promoter. The capacity of the whole-cell system to modify different hydroxysteroids was investigated, and NMR spectroscopy revealed that all substrates were selectively transformed into the corresponding 21-acetoxy derivatives. The biotransformation was optimized, and the reaction mechanism is discussed on the basis of a computationally modeled substrate docking into the crystal structure of CATI.

Master equation modeling of water dissociation in small ionic water clusters: Ag+(H2O) n , n = 4-6.

We model temperature-dependent blackbody infrared radiative dissociation (BIRD) rate coefficients of Ag+(H2O) n , n = 4-6, a system with loosely bound water molecules. We employ a master equation modeling (MEM) approach with consideration of absorption and emission of blackbody radiation, comparing single and multiple-well descriptions. The unimolecular dissociation rate coefficients are obtained using the Rice-Ramsperger-Kassel-Marcus (RRKM) theory, employing two approaches to model the sum of states in the transition state, the rigid activated complex (RAC) and the phase space limit (PSL) approach. A genetic algorithm is used to find structures of low-lying isomers for the kinetic modeling. We show that the multiple-well MEM approach with PSL RRKM in the All Wells and Transition States Are Relevant (AWATAR) variant provides a reliable description of Ag+(H2O) n BIRD, in agreement with previously published experimental data. Higher-lying isomers contribute significantly to the overall dissociation rate coefficient, underlying the importance of the multiple-well ansatz in which all isomers are treated on the same footing.

High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of Plasmodium falciparum.

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.

Batch tautomer generation with MolTPC.

Besides all their conformational degrees of freedom, drug-like molecules and natural products often also undergo tautomeric interconversions. Compared to the huge efforts made in experimental investigation of tautomerism, open and free algorithmic solutions for prototropic tautomer generation are surprisingly rare. The few freely available software packages limit their output to a subset of the possible configurational space by sometimes unwanted prior assumptions and complete neglection of ring-chain tautomerism. Here, we describe an adjustable fully automatic tautomer enumeration approach, which is freely available and also incorporates the detection of ring-chain variants. The algorithm is implemented in the MolTPC framework and accessible on SourceForge.

Resin acid conversion with CYP105A1: an enzyme with potential for the production of pharmaceutically relevant diterpenoids.

Cytochrome P450s are very versatile enzymes with great potential for biotechnological applications because of their ability to oxidize unactivated CH bonds. CYP105A1 from Streptomyces griseolus was first described as a herbicide-inducible sulfonylurea hydroxylase, but it is also able to convert other substrates such as vitamin D(3) . To extend the substrate pool of this interesting enzyme further, we screened a small diterpenoid compound library and were able to show the conversion of several resin acids. Binding of abietic acid, dehydroabietic acid, and isopimaric acid to the active site was assayed, and V(max) and K(m) values were calculated. The products were analyzed by NMR spectroscopy and identified as 15-hydroxyabietic acid, 15-hydroxydehydroabietic acid, and 15,16-epoxyisopimaric acid. As the observed products are difficult to obtain by chemical synthesis, CYP105A1 has proved to be a promising candidate for biotechnological applications that combine bioconversion and chemical synthesis to obtain functionalized resin acids.

CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase.

Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and ß-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and ß-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-ß-ionone and 13-hydroxy-nootkatone, were confirmed by (1)H and (13)C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K ( m ) and k (cat) values were calculated. The results of docking α-/ß-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.

Structural comparison of the cytochrome P450 enzymes CYP106A1 and CYP106A2 provides insight into their differences in steroid conversion.

Understanding the structural basis of the selectivity of steroid hydroxylation requires detailed structural and functional investigations on various steroid hydroxylases with different selectivities, such as the bacterial cytochrome P450 enzymes. Here, the crystal structure of the cytochrome P450 CYP106A1 from Priestia megaterium was solved. CYP106A1 exhibits a rare additional structural motif of a cytochrome P450, a sixth ß-sheet. The protein was found in different unusual conformations corresponding to both open and closed forms even when crystallized without any known substrate. The structural comparison of CYP106A1 with the previously investigated CYP106A2, including docking studies for both isoforms with the substrate cortisol, reveals a completely different orientation of the steroid molecule in the active sites. This distinction convincingly explains the experimentally observed differences in substrate conversion and product formation by the two enzymes.

Determining the degree of randomness of descriptors in linear regression equations with respect to the data size.

Linear regression equations suffer from the curse of dimensionality that leads to overfitting and accidental correlation, particularly for small data sets and when many variables are present. This can lead to cases where descriptors based on random numbers exhibit higher correlations than actual descriptors. In this study, it was therefore investigated how high the degree of accidental correlation of a single descriptor can be with respect to the number of observations. On the basis of computer simulations for data sizes ranging from 7 to 500 observations, a formula was derived that expresses the degree of randomness (in percent) of a chosen descriptor depending on its correlation coefficient and the size of the data set. This allows one to determine a cutoff for the correlation below which descriptors can be discarded due to a high risk of chance correlation. Doing so, the number of eligible variables for the regression analysis can be reduced substantially. Corresponding applications are reported for several QSAR data sets of various sizes.

Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine.

Saturation mutagenesis at seven first-sphere residues of the cytochrome P450 monooxygenase 154E1 (CYP154E1) from Thermobifida fusca YX was applied to construct a variant with only three substitutions that enabled the effective two-step synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine. A recombinant E. coli whole-cell system was essential for GC/MS based medium-throughput screening and at the same time facilitated the oxidation of the substrate (R)-ketamine at a higher scale for product isolation and subsequent NMR analysis.

Metabolism of oral turinabol by the human brain cholesterol 24-hydroxylase CYP46A1.

The human microsomal cytochrome P450 enzyme CYP46A1 plays a crucial role in cholesterol elimination from the brain. It performs a 24-hydroxylation of cholesterol and is of outstanding significance for memory and cognition. This study demonstrates the catalytic activity of human CYP46A1 towards an anabolic androgenic steroid, oral turinabol (dehydrochloromethyltestosterone, 4-chloro-17ß-dihydroxy,17α-methylandrosta-1,4-dien-3-one), which is a doping substance. CYP46A1 is the first human microsomal steroid-converting P450 showing activity towards this xenobiotic compound. Furthermore, the inhibitory effect of oral turinabol on the cholesterol conversion has been investigated in vitro demonstrating competition of the two substrates on the active site of CYP46A1 which might be of importance for potential pathogenic effects of oral turinabol. The conversion of oral turinabol was found to be selective resulting in the formation of only one product, as shown by HPLC analysis. To produce sufficient amounts of this product for NMR analysis, a system expressing human full-length CYP46A1 and CPR on a bicistronic vector was successfully developed realizing the selective cholesterol 24-hydroxylation in E. coli in mg amounts. Using this novel whole-cell system, the conversion of oral turinabol was performed and the product of this conversion by CYP46A1 was isolated and identified as 16ß-hydroxy oral turinabol by NMR.